The developmental cycle of Chlamydia trachomatis in McCoy cells treated with cytochalasin B.

The growth of a genital trachoma-inclusion conjunctivitis agent strain of Chlamydia trachomatis in McCoy cells treated with cytochalasin B was studied by quantitative infectivity estimations and by light and electron microscopy. Provided that infection of the monolayer was initiated by centrifuging the infectious particles on to the cells before incubation, this chlamydial strain grew as fast and to as high a titre [approximately 10(7) inclusion-forming units (i.f.u.) per culture] as those chlamydiae which infect cell cultures in vitro without centrifugation. Each i.f.u. inoculated yielded approximately 600 i.f.u., and extracellular infectivity was detected soon after intracellular infectivity appeared. Inclusions were recognized by fluorescent antibody staining techniques early in the developmental cycle when cultures were not infectious and when only reticulate bodies were seen by electron microscopy. Inclusions were recognized in Giemsa-stained preparations examined by dark ground microscopy only when elementary bodies appeared in the inclusions. Iodine staining was not a reliable indicator either of the number of inclusions present or of their infectivity.     

Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells

Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia.     

Trifluoperazine inhibits the infectivity of Chlamydia trachomatis for McCoy cells

Abstract Trifluoperazine (TFP), an inhibitor of the Ca²⁺-binding protein calmodulin, was used to study the infectivity of Chlamydia trachomatis for McCoy cells. TFP inhibited the number of chlamydial inclusions and the chlamydia-dependent amino acid incorporation when added within 9 h after inoculation with chlamydiae. However, TFP did not affect the attachment of chlamydiae to the cells or the protease-removable fraction of cell-bound chlamydiae.
These results suggest that an early step in the intracellular development of chlamydiae, partly coinciding with the elementary body-reticulate body conversion, is sensitive to TFP and that clathrin coats are not crucial in the ingestion of chlamydiae by McCoy cells.     

Division and transmission of inclusions of Chlamydia trachomatis in replicating McCoy cell monolayers

Abstract When Chlamydia trachomatis serotype E was grown in monolayers of replicating McCoy cells, dividing inclusions were seen by indirect immunofluorescence. Transmission of inclusions occurred within the McCoy cell population, so that clusters of inclusions in adjacent cells had formed by 72 h. Inclusion division and transmission may provide an important mechanism for persistence of naturally occurring chlamydial infections.     

Effect of Ionizing Irradiation on Susceptibility of McCoy Cell Cultures to Chlamydia trachomatis

The effect of graded doses of irradiation (cobalt-60) on the morphology of McCoy cells was analyzed, and 4,000 to 5,000 r was selected as a satisfactory dose for production of giant cells. The susceptibility of radiation-induced giant cells to chlamydial infection was compared with that of nonirradiated cells by using three strains of Chlamydia trachomatis and one of C. psittaci. Monolayers of giant cells were more susceptible than normal McCoy cells as indicated by (i) greater numbers of inclusions (four- to eightfold) per unit area of monolayer, (ii) larger inclusions (fourfold greater in area), (iii) higher infective titers (1 log or more greater) of harvested cells, and (iv) greater ease of promoting a second cycle of growth. Graded doses of irradiation were applied also to mouse fibroblast (L) cells, and a similar increase in susceptibility to chlamydial infection was noted. It is concluded that giant cells produced by irradiation possess advantages over nonirradiated cells in culture for growth of Chlamydia.     

Immunoelectron microscopic localization of chlamydial lipopolysaccharide (LPS) in McCoy cells inoculated with Chlamydia trachomatis.

Interactions between Chlamydia trachomatis, host cells, and the immune system are believed to involve lipopolysaccharide (LPS). We used immunogold techniques to study the distribution of chlamydial LPS in cultured cells infected with C. trachomatis LGV-L1. McCoy cells inoculated with C. trachomatis were cultured and then fixed and embedded in situ with acrylic resins. Sections were immunolabeled with a protein A-gold method using antisera to the genus-specific, periodate-sensitive epitope on chlamydial LPS. Pre-embedding immunogold labeling on permeabilized cells was also done. By post-embedding methods, labeling for LPS was equally abundant over the outer membranes of elementary (EB) and reticulate bodies (RB). By post-embedding labeling, the sub-surface side of the EB outer membrane was more heavily labeled than the surface side. By pre-embedding labeling, LPS was found to be less abundant on the surface of EBs than RBs. Labeling for LPS was found over apparent lysosomes in McCoy cells and over electron-dense blebs on or near the surface of the plasma membranes of McCoy cells. These results indicate that the concentration of LPS in chlamydial membranes is constant during development but that with development its location changes from being mostly cell-surface to sub-surface. These results show that the post-embedding immunogold technique can be a useful approach for the cell culture-based study of chlamydial LPS.     

Use of ultrasound to increase infection of McCoy cell monolayers by Chlamydia trachomatis strain

An ultrasound cell disrupter with a cooled cup tip was used to increase rapidly Chlamydia trachomatis infection in vitro. After three growth cycles of the NI-1 strain (serovar E), the pulsed ultrasound use enhanced the number of infected McCoy cells by approximately 12-times, as compared with control; and 8.8-times over the shaking with glass beads and centrifugation technique. After three growth cycles of the fast-growing LB-1 strain (serovar L2), the enhancement was by 15 and 10.8 respectively. Consequently, ultrasound treatment with a cooled cup tip can offer a working standard procedure to increase rapidly the number of cells infected with Chlamydia.     

Cytochalasin B and Release of Growth Hormone

MICROTUBULES are believed to be involved in emiocytosis, in which secretory granules migrate and fuse with the cell membrane, thereby liberating the granule contents into the extracellular space. Important evidence for this is that the release of insulin from pancreatic islets of Langerhans^1,2, histamine from mast cells³ and from leucocytes⁴ and catecholamines from the adrenal medulla⁵ is inhibited by colchicine, which, at lower concentrations, disrupts microtubular systems⁶. Although the function of microtubules in the secretory process is far from clear, it has been postulated that they are part of a contractile system which could move granules to the cell surface, or could be involved in the final release mechanism⁵. If contractile proteins do play a fundamental part in secretion in the four systems investigated, they would also be expected to be involved in other secretory systems. However, stimulation of growth hormone release in vitro is inhibited by colchicine only at the high concentration of 5 mM, at which concentration basal release is doubled by the drug⁷. This relative insensitivity to colchicine could be reconciled with an involvement of contractile proteins in secretion if microfilaments, rather than microtubules, were important in the release of growth hormone. Microfilaments are not affected by treatment with colchicine⁸ but may be disrupted by cytochalasin B⁹ and the effect of this drug on in vitro secretion of ox growth hormone in response to Ba²⁺, high extracellular K⁺ and prostaglandin E₂ has therefore been tested.     

Processing of McCoy cell cultures infected with Chlamydia trachomatis: sequential isolation of chlamydial elementary bodies and lipopolysaccharide

Abstract Triton X-100 (TX100) enhances the liberation of chlamydial elementary bodies (EB) from host cells and dissolves the host cell membrane. In the presence of TX100 only differential centrifugation is needed to isolate reasonably pure EBs. The remaining high-speed supernatant still contains a large part of the chlamydial lipopolysaccharide (LPS), which can be isolated with the standard phenol-chloroform-petroleum ether extraction.     

Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K⁺ through glibenclamide-sensitive K⁺ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, and it is the leading cause of preventable blindness in the world (1–5). Untreated, C. trachomatis infection in women can cause pelvic inflammatory disease, which can lead to infertility and ectopic pregnancy because of scarring of the ovaries and the Fallopian tubes (6). Infection by the lymphogranuloma venereum (LGV)² strain of C. trachomatis, which has become more common in North America and Europe (7, 8), is characterized by swelling and inflammation of the lymph nodes in the groin (9).Chlamydiae are intracellular pathogens that preferentially infect epithelial mucosa and have a biphasic infection cycle (10). A metabolically inactive form, the elementary body, infects the epithelial host cells through entry vesicles that avoid fusion with host cell lysosomes and develop into a membrane-bound inclusion (11–13). Despite their intravacuolar localization, chlamydiae are still able to acquire nutrients from the host cell and interact with host-cell signaling pathways (13–23). Within a few hours, the elementary bodies differentiate into larger, metabolically active reticulate bodies, which proliferate but are noninfectious. Depending on the strain of C. trachomatis, the reticulate bodies transform back into elementary bodies after 1–3 days and are released into the extracellular medium to infect other cells (11, 24, 25). Chlamydial species possess a type III secretion (T3S) system that secretes bacterial virulence factors into host cell cytosol and may control interactions between the inclusion and host-cell compartments (26).Long before the adaptive immune response is activated, infected epithelial cells produce proinflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (27), which recruit neutrophils to the site of infection and activate other immune effector cells. However, in many cases the immune system fails to clear the infection, and the chronic release of cytokines becomes a major contributor to the scarring and damage associated with the infection (28–30).The innate immune response during C. trachomatis infection is initiated by chlamydial pathogen-associated molecular patterns, including lipopolysaccharides, which bind to pattern recognition receptors such as Toll-like receptors and cytosolic NOD-like receptors (NLRs), ultimately promoting pro-inflammatory cytokine gene expression and secretion of the cytokine proteins (31–37). However, secretion of the key pro-inflammatory cytokine IL-1β is tightly regulated (38). First, pro-IL-1β is produced following activation of pattern recognition receptor, and the precursor is then cleaved into the mature form by the pro-inflammatory cysteine protease, caspase-1 (also known as interleukin-1 converting enzyme or ICE). The mechanism by which caspase-1 is activated in response to infection or tissue damage was found to be modulated by a macromolecular protein complex termed the “inflammasome,” which consists of an NLR family member, an adaptor protein (apoptosis-associated speck-like protein containing a caspase activation recruitment domain or ASC), and an inactive caspase-1 precursor (pro-caspase-1) (39, 40). Previous studies demonstrated that IL-1β is produced in response to chlamydial infection in dendritic cells, macrophages, and monocytes (41–44). Moreover, C. trachomatis or Chlamydia caviae infection activates caspase-1 in epithelial cells or monocytes (43, 45, 46). However, whether caspase-1 activation during chlamydial infection requires the formation of an inflammasome remains unclear.Previous studies have shown that different pathogens can cause inflammasome-mediated caspase-1 activation in macrophages and monocytes (47). However, epithelial cells lining mucosal surfaces are not only the preferred target for chlamydial infection and other intracellular pathogens but also play an important role in early host immune response to infection by secreting proinflammatory cytokines and chemokines (27). Although epithelial cells are not known to secrete large amounts of IL-1β, inflammasome-dependent caspase-1 activation in epithelial cells is known to contribute to lipid metabolism and membrane regeneration in epithelial cells damaged by the membrane-disrupting toxin, aerolysin (48). As lipids are sorted from the Golgi apparatus to the chlamydial inclusion (13, 15, 49), we therefore investigated whether C. trachomatis induces caspase-1 activation in epithelial cells via the assembly of an inflammasome. We demonstrated that C. trachomatis-induced caspase-1 activation is mediated by an inflammasome containing the NLR member, NLRP3. Several studies have demonstrated the involvement of T3S apparatus in inflammasome-mediated caspase-1 activation by different pathogens in macrophages and monocytes (50–56). Therefore, we further investigated the mechanism by which C. trachomatis triggers the formation of the NLRP3 inflammasome. Our results showed that metabolically active chlamydiae, relying on their T3S apparatus, cause K⁺ efflux, which in turn leads to formation of reactive oxygen species (ROS) and ultimately NLRP3-dependent caspase-1 activation. Epithelial cells do not typically secrete large amounts of IL-1β; instead, caspase-1 activation in cervical epithelial cells contributes to development of the chlamydial inclusion.     

Factors affecting the adhesion and uptake of an oculo-genital strain of Chlamydia trachomatis to McCoy cells

Abstract We have studied adhesion and uptake of C. trachomatis serovar E in McCoy cells under various infection conditions. Adhesion and uptake of chlamydiae was completed about 3 h after the initiation of stationary infection at 37°C, but ingestion of cell membrane-attached organisms was finished within 0.5 h at 37°C. Reincubated chlamydiae, not attached after 3 h at 37°C, attached readily to fresh McCoy cell monolayers, but to a lesser extent than the original inoculum. Our results indicate that the lack of further attachment after 3 h incubation at 37°C under stationary infection conditions has complex causes, involving both host cell and parasite. Centrifugation did not affect the uptake of chlamydiae already bound to the cell membrane, suggesting that the uptake phase of C. trachomatis serovar E by McCoy cells is unaffected by centrifugation.     

Growth of animal cells on microbeads. II. Estimation of biomass in situ and observation of cytopathic effects after infection of McCoy cells with Chlamydia trachomatis

The Channelyzer C256 together with the ZB Coulter Counter have been used to study in situ the growth kinetics of McCoy cells attached on Cytodex 3 microbeads. The number of cells per microbead varied linearly with the average size of the covered microbeads, showing that electronic particle sizing can be used to measure biomass or cell numbers. As the cells became confluent, an increase in cell volume with little accompanying increase in cell number was detected. The technique also enables the experimenter to follow in situ the development of cytopathic effects resulting from the infection by pathogens such as Chlamydia trachomatis serotype L1.     

Effects of Cytochalasin B on Muscle Cells in Tissue Culture

THE antibiotic cytochalasin B induces the formation of binucleated and multinucleated cells by preventing cytokinesis¹ and the cleavage furrow filaments in sea urchin eggs, 50-100 Å in diameter, disappear in the presence of this compound². It has a similar effect on the fine filaments found in embryonic pancreatic cells³ and inhibits cell migration¹. Many kinds of cells displaying amoeboid movements have been reported to have 50-100 Å filaments^4–6. Ishikawa et al. have demonstrated that 60–80 Å filaments in the cortex of various tissue cells bind heavy-meromyosin in a manner identical to that of actin filaments, which are also 60–80 Å in diameter⁷. Many investigators have referred to these thin filaments as actin or actin-like and assumed them to be responsible for, or associated with, cellular movements^8,9, contraction^10,11 and cytokinesis^12,13.     

genistein对沙眼衣原体感染细胞信号转导的影响

研究酪氨酸激酶抑制剂 genistein对IFN γ诱导沙眼衣原体感染细胞内信号转导的影响。以IFN γ作用于沙眼衣原体 (K血清型 )感染的McCoy细胞 ,用 genistein阻断IFN γ对沙眼衣原体感染细胞的作用。用台盼蓝染色法检测沙眼衣原体感染细胞存活率。用免疫印迹法检测蛋白酪氨酸激酶磷酸化及JAK1/STAT1活化的改变。结果显示 :genistein可拮抗IFN γ降低沙眼衣原体感染细胞存活率的作用 ,且具有剂量依赖效应。genistein可抑制IFN γ诱导的沙眼衣原体感染细胞蛋白酪氨酸激酶磷酸化及JAK1/STAT1活化 ,此作用且与genistein作用的剂量有关。     

Chlamydia trachomatis in cell culture

Summary Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S₃, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S₃ and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO₆₀ McCoy and CO₆₀ BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO₆₀ BHK-21 Lister than in CO₆₀ McCoy. This research was supported by a grant from the National Eye Institute (EI-00812-08), and by the Arabian American Oil Company. The paper is dedicated to the memory of Francis B. Gordon, who pioneered research methods for the cultivation of trachoma and inclusion conjunctivitis (TRIC) agents in cell culture. Dr. Gordon patiently studied tables and photographs which accompany this text when he visited our laboratory on the day prior to his sailing to England on the ill-fated voyage in which he and Mrs. Gordon perished (October 1973).     

用细胞与鸡胚二步法培养眼-泌尿生殖道衣原体的研究

将眼－泌尿生殖道衣原体在ＭｃＣｏｙ细胞中扩大培养至５０％以上细胞形成包涵体以后,用细胞培养物接种７日龄鸡胚卵黄囊,使其在鸡胚卵黄囊中生长。用此二步法联合制备衣原体抗原,克服了单独用细胞法和卵黄囊法的不足,可在短时间里获得较多的抗原。     

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.